Immunogenic Activity of a Ribosomal Fraction Obtained from Mycobacterium tuberculosis

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Abstract
The highly immunogenic particulate fraction obtained from mechanically ruptured cells of the H37Ra strain of M. tuberculosis was suspended and centrifuged at 20,360 x g. The supernatant liquid from this centrifugation was centrifuged at 56, 550 x g to remove the larger particles, and the supernatant liquid from this was centrifuged at 144,000 X g to obtain a ribosomal fraction. The sediments from the first two centrifugations were highly immunogenic, but the ribosomal fraction showed only slight capacity to immunize mice. However, when the ribosomal fraction was mixed with Freund''s incomplete adjuvant, the immunogenic activity was equivalent to the particulate fraction from which it was prepared. To test the hypothesis that some membranous substance in the particulate fraction was acting as an adjuvant for the smaller particles in the ribosomal fraction, portions of the particulate fraction were treated separately with each of the membrane-disruptingagents, sodium-deoxycholate, sodium-lauryl-sulfate, and 1 M sodium-chloride. The treated materials were then centrifuged at 144,000 X g, and the sediments were tested for immunogenicity both with and without the addition of Freund''s incomplete adjuvant. Without the adjuvant, the immunizing activities were very weak or absent; with the adjuvant, they were equivalent to that of the particulate fraction from which they were prepared. Other factors which have been found to damage or destroy membranes, such as freezing and thawing and heat, also significantly decreased the immunogenic activity of the particulate fraction unless it was incorporated into Freund''s incomplete adjuvant. The larger particles which sedimented at 56,550 X g were also treated with sodium-lauryl-sulfate and sodium-chloride. Again, immunogenicity was greatly reduced but was fully restored by use of Freund''s incomplete adjuvant. The data suggest, then, that the immunizing component of the particulate fraction is a substance (ribosomal?) which sediments at 144,000 X g, but for maximal immunizing activity a labile, possibly membranous, moiety of the mycobacterial cell, which has the properties of an adjuvant, is required.